Several genetically engineered strains of Candida tropicalis have been used in fermentations for the bioconversion of the 18-carbon fatty acid known as oleic acid to 18-carbon dicarboxylic acid. When grown on fatty acids, wild-type C. tropicalis converts long chain fatty acids to acetyl CoA by a process known as β-oxidation, which is the sequential catabolism of 2-carbon length fragments of a fatty acid to acetyl CoA. β-Oxidation is thus named because the initial oxidative attack occurs at the second carbon atom from the carboxylic group. C. tropicalis can also catabolize fatty acids through an ω-oxidation pathway in which only the terminal methyl carbon is oxidized to a carboxylic acid, yielding a dicarboxylic acid. In ω-oxidation, the fatty acid is converted to dicarboxylic acid along a three-step pathway beginning with the oxidation of the terminal methyl group to an alcohol. This step is catalyzed by the hydroxylase complex that contains both the cytochrome P450 monooxygenase (CYP) and cytochrome P450 reductase (NCP) proteins. The alcohol is then converted to an aldehyde by fatty alcohol oxidase (FAO) and then to the dicarboxylic acid by an aldehyde dehydrogenase. The desired product is the long-chain dicarboxylic acid. A fatty alcohol oxidase is distinguished from an alcohol oxidase in its chain length specificity. Alcohol oxidases in general are specific for methanol but can sometimes oxidize alcohols up to C4. Fatty alcohol oxidases generally do not oxidize alcohols with chain lengths less than eight.
In wild-type Candida tropicalis, β-oxidation consumes fatty acids much faster than the ω-oxidation pathway can oxidize them. However, by inactivating the POX 4 and POX 5 genes, which gene products are responsible for the initiation of β-oxidation, such genetically engineered C. tropicalis strains preferentially shunt fatty acids into the ω-oxidation pathway. The base strain used for the development of various gene-amplified strains is H5343, which is C. tropicalis strain 20336 (American Type Culture Collection) with both POX 4 and POX 5 genes inactivated by insertional inactivation. The primary strain used in larger-scale production fermentations is HDC23-3, which is derived from H5343, but also has the CYP52A2 (a cytochrome P450 monooxygenase) gene amplified. The hydroxylase complex is responsible for catalyzing the first step in ω-oxidation, which is considered the rate-limiting step. Amplification of the CYP52A2 and NCP genes help to overcome this rate-limitation, but then the next bottleneck becomes the conversion of the alcohol to the aldehyde by the FAO enzyme. During fermentations with HDC23-3, it has been discovered that a small amount (ca. 0.5% w/w in broth) of ω-hydroxy fatty acid (HFA) accumulates during the fermentation. This partial oxidation product interferes with later purification steps and causes lower overall yields. There is an additional need therefore, for reducing the bottleneck in the conversion of the alcohol to an aldehyde during the second step of the ω-oxidation pathway.
A small number of fatty alcohol oxidases have been described in the scientific literature in various yeasts, examples of which are Candida tropicalis (1, 2, 3, 4), Candida maltosa (5,6), Candida cloacae (4), Torulopsis candida (7), Candida (Torulopis) bombicola (8), and Candida (Torulopsis) apicola (9). The FAO was purified from the hexadecane-grown yeast, T. candida (7) and described as a tetramer (mw 290 kD) with subunit mol. wt. of 75 kD. It has a pH optimum of 7.6 and oxidizes higher alcohols with a carbon chain length of C4 to C16. Hexadecane-grown C. bombicola (8) apparently has two different alcohol oxidase activities, one with an optimal chain length specificity of 10 for n-alcohols and another with an optimal chain length specificity of 14. The FAO from C. maltosa (6) catalyzes the oxidation of 1-alkanols (C4 to C22) with highest activity utilizing 1-octanol. It also oxidizes 2-alkanols (C8 to C16). α,ω-Alkanediols, ω-hydroxypalmitic acid, phenylalkanols and terpene alcohols were all found to be substrates for the FAO, but at fairly low rates of oxidation. The oxidation of 2-alkanols is stereoselective for the R(−) enantiomers only.
The FAO from C. tropicalis (ATCC 20336) grown on hexadecane was first described by Kemp et al. in 1988 (1). It was found to oxidize 1-alkanols from C4 to C18, but has a maximal activity with dodecanol. It was found to oxidize 16-hydroxypalmitate but not 12-hydroxylaurate. The FAO was later purified (3) and was shown to be a dimer (mw=145 kD) with subunit molecular weight of 68–72 kD. The purified enzyme showed similar substrate specificity as described previously, but demonstrated additional activity with 12-hydroxylaurate and 2-dodecanol. The enzyme was found to be a light sensitive flavoprotein, but the identity of the flavin was not known.(10).
Recently two FAO genes from C. cloacae and one FAO gene from C. tropicales were cloned and the DNA sequences determined (4, 12). The open reading frame (ORF) for FAO1 and FAO2 from C. cloacae were 2094 bp and 2091 bp, respectively. The ORF for FAOT from C. tropicalis was 2112 bp. FAOT shares 60.6% and 61.7% nucleotide identities and 74.8% and 76.2% amino acid sequence similarities with C. cloacae FAO1 and FAO2, respectively. The FAO1 gene but not the FAO2 gene has been successfully cloned and expressed in Escherichia coli. 
The present invention provides FAO genes from C. tropicalis and compositions and methods employing the FAO genes. The compositions and methods are useful for increasing FAO activity during the second step of omega-oxidation of fatty acids and ultimately result in an increase in diacid productivity.